The field of the invention is antimicrobial therapy for bacterial infection.
The respiratory tract of mammals constitutes a barrier between the internal and external milieu. This barrier serves as a first line of defense against invasion by microbial agents. In humans, this defense comprises physical barriers such as mucocillary clearance, biochemical defenses including complement, lysozyme and antibodies, and cells which are capable of generating an inflammatory immune response such as alveolar macrophages (Canto et al.: In: Pulmonary Infections and Immunity, H. Chmel et al., Eds., Plenum, New York, 1994, pp.1-27).
One component of this line of defense is a family of peptides known as defensins. Mammalian defensins are small antimicrobial peptides (3.5-4.5 kDa) that are characterized by the presence of six cysteines which form three disulfide bonds whose ordered array determines whether these proteins are classified as xcex1 or xcex2 defensins. The xcex1 defensins are found in granulozytes (Ganz et al., 1995, Pharm. Therap. 66:191-205) and Paneth cells of the small intestine (Jones et al., 1992, J. Biol. Chem. 267:23216-23225; Mallow et al., 1996, J. Biol. Chem. 271:4038-4045) in various species, whereas xcex2-defensins occur in leucogytes of cattle (Selsted et al., 1993, J. Biol. Chem. 268:6641-6648; Tang et al., 1993, J. Biol. Chem. 268:6649-6653) and fowl (Harwig et al., 1994, FEBS Letters 342:281-285; Evans et al., 1994. J. Leuc. Biol. 56:661-665), where they contribute to host defense system of mucosal surfaces. Lingual and tracheal antimicrobial peptides are xcex2 defensins expressed in bovine epithelial cells of tongue and trachea (Diamond et al., 1991, Proc. Natl. Acad. Sci. USA 88:3952-3956; Diamond et al., 1993, Proc. Natl. Acad. Sci. USA 90:4596 -4600; Schonwetter et al., 1995, Science 267:1645-1648).
The first xcex2 defensin isolated from humans, the human xcex2-defensin 1 (hBD-1), is found in salivary glands, airways, prostate and placenta among other tissues (Bensch et al., 1995, FEBS Letters 368:331-335; Goldman et al., 1997, Cell 88:553-560; McCray et al., 1997, Am. J. Respir. Cell. Mol. Biol. 16: 343-349; Zhao et al., 1996, FEBS Letters 396: 319-322). The expression of mRNA specifying hBD-1 is detected in the colon, small intestine, kidney, prostate, liver and pancreas.
hBD-1 was originally isolated from the hemofiltrate of dialysis patients (Bensch et al., supra). A cDNA sequence specifying only 38 amino acids of mature hBD-1 was identified by Bensch et al. (supra). FIG. 1 illustrates this partial cDNA sequence. The upper line represents the amino acid sequence in single letter code. The primers BDNT and UNIP-2 used for cloning are represented as arrows. The first seventeen nucleotides originate from the degenerate primer BDNT so all possible nucleotides for the third codon position are listed in the sequence (Y=C or T).
Cystic fibrosis (CF) is a multisystem obstructive disease characterized by chronic infection of the airway (Decker et al., 1993, In: Cystic Fibrosis, pp. 193-218, P. B. Davis, Ed., Dekker, New York). The disease is caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR), leading to altered Na+ and Clxe2x88x92 transport and abnormal composition of airway surface fluid (Collins et al., 1992, Science 256:774-779; Joris et al, 1993, Am. Rev. Respir. Dis. 148:1633-1637; Quinton, 1990, FASEB J. 4:2709-2717).
Many hypotheses have been set forth to explain the propensity of the CF airway to become infected (Lehrer et al., 1983, Infect. Immun. 42:10-14; Zasloff, 1987, Proc. Natl. Acad. Sci. USA 84-5449-5453; Konstan et al., 1993, In: Cystic Fibrosis, pp.219-276, P. B. DAVIS, Ed, Dekker, New York; Imundo et al., 1995, Proc. Natl. Acad. Sci. USA 92:3019-3023; Pier et al., 1996, Science 271:64-67). Despite this, the causal chain of events leading from genetic lesions in CFTR to the devastating lung infections suffered by CF patients remains unclear. It has been proposed that dysfunction of airway antimicrobial peptides may be a critical contributing factor to lung infection (Lehrer et al., 1983, supra; Zasloffet al., 1987, supra).
Very recently, antimicrobial activity has been detected in the surface fluid of healthy cultured human airway epithelium (Smith et al., 1996, Cell 85:229-236). Although hBD-1 is widely expressed, antimicrobial activity was absent in the high salt environment of cystic fibrosis epithelium. The activity of a defensin-like molecule returned when the salt concentration was lowered. Cystic fibrosis associated infection may be limited to the respiratory system due to its direct association with the external environment. In addition to the high salt which is the result of defective chloride transport, evaporation of fluid from the airway surface may further concentrate the luminal airway fluid. Both the increase in salt and evaporation of fluid may lower the effects of hBD-1 in CF patients.
There remains a need for antimicrobial therapy directed against bacterial infections in patients having cystic fibrosis and other infectious diseases. There also remains a need for an isolated full length cDNA precursor sequence for hBD-1, that is, a defensin molecule which can be used for the prevention and treatment of infection and for other therapeutic ends. There further remains a need for the development of suitable animal models for the study of the role of defensins in protection against microbial disease and for the discovery of therapeutic agents to combat such disease.
The invention relates to a cell comprising an isolated nucleic acid encoding human beta defensin-1.
The invention also relates to a cell comprising an isolated nucleic acid encoding rodent beta defensin-1. In one aspect, the rodent defensin is mouse beta defensin-1. In addition, the nucleic acid may further comprises a promotor/regulatory sequence positioned at the 5xe2x80x2 end of the human beta defensin-1 or the rodent beta defensin-1.
The invention also includes a vector comprising an isolated nucleic acid encoding human beta defensin-1. In one aspect, the vector may be selected from the group consisting of a plasmid, a virus and a non-viral vector and in another aspect, the vector may be suspended in a pharmaceutical composition. In yet another aspect, the isolated nucleic acid in the vector further comprises a promoter regulatory sequence positioned at the 5xe2x80x2 end of the human beta defensin.
There is also included in the invention a vector comprising an isolated nucleic acid encoding rodent beta defensin-1. In one aspect of this aspect of the invention, the defensin is mouse beta defensin-1. In another aspect, the vector is selected from the group consisting of a plasmid, a virus and a non-viral vector, and in yet another aspect, the isolated nucleic acid further comprises a promotor/regulatory sequence positioned at the 5xe2x80x2 end of the rodent beta defensin-1.
Also included in the invention is an isolated nucleic acid encoding human beta defensin-1. Preferably, the isolated nucleic acid is cDNA and more preferably, the isolated nucleic acid is the sequence shown in FIG. 2 [SEQ ID NO:3].
In one aspect, the isolated nucleic acid further comprises a promoter/regulatory sequence positioned at the 5xe2x80x2 end of the coding region of the human beta defensin-1.
The invention further includes an isolated nucleic acid encoding mouse beta defensin-1. Preferably, the isolated nucleic acid is cDNA. More preferably, the isolated nucleic acid is the sequence shown in FIG. 12 [SEQ ID NO:6].
In one aspect, the isolated nucleic acid may further comprise a promoter/regulatory sequence positioned at the 5xe2x80x2 end of the mouse beta defensin-1.
The invention also includes an isolated nucleic acid encoding a saltinsensitive mammalian beta defensin-1. The mammalian beta defensin-1 may be selected from the group consisting of human beta defensin-1 and mouse beta defensin-1 and the isolated nucleic acid may further comprise a promoter/regulatory sequence positioned at the 5xe2x80x2 end of the mammalian beta defensin-1.
Also included in the invention is an isolated nucleic acid encoding a mammalian beta defensin-1 having enhanced antimicrobial activity when compared with a wild type mammalian defensin-1 counterpart. In one aspect, the mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1, and in another aspect, the isolated nucleic acid further comprises a promoter/regulatory sequence positioned at the 5xe2x80x2 end of the mammalian beta defensin-1.
There is also included in the invention a salt insensitive mammalian beta defensin-1, which may be selected from the group consisting of human beta defensin-1 and mouse beta defensin-1.
In addition, the invention includes a mutated mammalian beta defensin-1 having enhanced antimicrobial activity when compared with a wild type mammalian defensin-1 counterpart. In one aspect of this aspect of the invention, the mutated mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1.
There is also provided in the invention a method of enhancing antimicrobial activity in a tissue sample comprising adding to the sample a mammalian beta defensin-1. In one embodiment, the tissue sample is selected from the group consisting of a mammalian lung tissue sample, a mammalian skin tissue sample and a mammalian blood tissue sample. In another embodiment, the mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1.
In one aspect, the mammalian beta defensin-1 is added to the tissue sample in vivo in a mammal.
In another aspect, the tissue sample is a lung tissue sample and the mammalian beta defensin is human beta defensin-1 which is added to the lung tissue sample by means of a nebulizer or a bronchoscope.
In yet another aspect, the tissue sample is a lung tissue sample and the mammalian beta defensin is human beta defensin-1 which is added to the lung tissue sample in the form of a vector comprising an isolated nucleic acid encoding the human beta defensin-1. When the vector is administered to the human the human beta defensin-1 is expressed therefrom to effect addition of the human beta defensin-1 to the lung tissue sample. In one aspect, the human has a respiratory disease which is preferably emphysema or cystic fibrosis. However, when the respiratory disease is cystic fibrosis, the vector encoding human beta defensin may be salt-insensitive or may have added thereto a compound capable of absorbing salt.
In yet another aspect, the human has a respiratory disease which predisposes the human to pulmonary microbial infection. Preferably, the respiratory disease is emphysema or cystic fibrosis. However, when the respiratory disease is cystic fibrosis, the human beta defensin may be salt-insensitive or may have added thereto a compound capable of absorbing salt.
There is further included in the invention a method of treating a human having a respiratory disease which predisposes the human to pulmonary microbial infection, the method comprising administering to the lungs of the human a pharmaceutical composition comprising human beta defensin-1.
In addition, the invention relates to a method of treating a human having a respiratory disease which predisposes the human to pulmonary microbial infection, the method comprising administering to the lungs of the human a pharmaceutical composition comprising an isolated nucleic acid encoding human beta defensin-1 wherein the human beta defensin-1 is expressed from the isolated nucleic acid in cells of the lungs thereby treating the human.
The invention also includes a method of treating a human having a pulmonary infection, the method comprising administering to the lungs of the human a pharmaceutical composition comprising human beta defensin-1.
There is also provided a method of treating a human having a pulmonary microbial infection, the method comprising administering to the lungs of the human a pharmaceutical composition comprising an isolated nucleic acid encoding human beta defensin-1 wherein the human beta defensin-1 is expressed from the isolated nucleic acid in cells of the lungs thereby treating the human.
In addition, there is provided a method of treating a human having a microbial infection of the skin comprising administering to the skin of the human a composition comprising human beta defensin-1.
Further, there is included a topical composition for administration to the skin of a mammal comprising mammalian beta defensin-1 suspended in a pharmaceutically acceptable carrier. In one aspect, the mammalian beta defensin is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1
Also provided is a pharmaceutical composition comprising human beta defensin-1.
In addition, the invention includes a transgenic mammal comprising an isolated nucleic acid encoding human beta defensin-1.
A method of synthesizing human beta defensin-1 using solid phase 9-Fluorenylmethyloxycarbonyl synthesis is also included. The method comprises regioselective formation of Cys5-Cys34, Cys12-Cys27 and Cys17-Cys35 by protecting Cys5-Cys34 with trityl (triphenylmethyl), protecting Cys12 and Cys27 with Acm (acetamidomethyl), and protecting Cys17 and Cys35 with MOL (p-methoxy benzyl).